Unknown Circovirus in Immunosuppressed Patient with Hepatitis, France, 2022

Hepatitis of undetermined origin can be caused by a wide variety of pathogens, sometimes emerging pathogens. We report the discovery, by means of routine shotgun metagenomics, of a new virus belonging to the family Circoviridae, genus Circovirus, in a patient in France who had acute hepatitis of unknown origin.

Hepatitis of undetermined origin can be caused by a wide variety of pathogens, sometimes emerging pathogens. We report the discovery, by means of routine shotgun metagenomics, of a new virus belonging to the family Circoviridae, genus Circovirus, in a patient in France who had acute hepatitis of unknown origin. markers of autoimmune hepatitis, and withdrawal or diminution of potentially hepatotoxic treatments had no effect on cytolysis. Aminotransferase levels started to decrease spontaneously 7 weeks after admission. SMg testing was prescribed to identify a potential treatable cause of this acute hepatitis. The patient expressed no opposition to the use of her data and samples for this purpose.
The SMg technique has already been described (2)(3)(4). We performed preextraction mechanical, enzymatic, and chemical actions before extracting both DNAs and RNAs using a DSP DNA Midi Kit on a QiaSymphony device (both QIAGEN, https://www.qiagen.com/us). We generated DNA libraries using a Nextera XT kit nd generated RNA libraries using a TruSeq Total RNA kit (both Illumina, https://www.illumina.com). We sequenced these libraries using NextSeq 500/550 High Output Kit v2.5 300 Cycles (Illumina). We performed metagenomics data analysis using MetaMIC software (https://gitlab.com/mndebi/metamic). The software filters out poor-quality data, identifies sequences by comparison with an nucleotide-based database, reduces background noise by comparison with environmental controls, and establishes a report on the presence or absence of bacteria, viruses, fungi, and parasites.
We performed data reanalysis for genome reconstruction and phylogenetic analysis. We assembled viral DNA sequences and RNA transcripts by using Metaspades 3.15.3 software (5). We assembled contigs by means of iterative in-house scripts, gradually replacing the closest reference viral sequences by the patient's sequences. We checked the consensus sequence by realigning the reads with bwa-mem 0.7.17-r1188 software (https://github.com/lh3/bwa) and by manual checking using the IGV 2.9.4 tool (https://software.broadinstitute.org/software/igv/). We performed phylogenetic analysis using a library of the replicase region and full-length Circovividae genome sequences (6), supplemented by the sequences closest to the newly identified virus found using BLASTn (https://blast.ncbi. nlm.nih.gov/Blast.cgi?PROGRAM=tblastn&PAGE_ TYPE=BlastSearch&LINK_LOC=blasthome) and the nucleotide database from GenBank, and MUSCLE alignment (7)  The viral genome sequence of 2,021 nt could be reconstructed (GenBank accession no. ON526744) (Figure, panel A). The origin of replication located in the AGTATTAC sequence had 1 nucleotide deletion compared with other circoviruses (Figure, panel A). We identified the 2 major circovirus open reading frames (ORFs), starting at positions 140 (replicase, ORF1/rep, sense) and 2,013 (capsid protein, ORF2/cap, antisense), as well as sense ORF3, starting at position 82 ( Figure, panel A). The 6 regions described as conserved within the rep region were present, identical to other species from the same genus (Figure, panel A).
By phylogenetic analysis, the new C. parisii clustered with other circoviruses, on the same branch as recently described wolverine circovirus (8), rodent circovirus (9), and Porcine circovirus 3. It was related to another branch containing bat circovirus ( Figure, panel B). The genetic distances between C. parisii and other circoviruses were of the same order as those between different circovirus species.
The presence of the virus was confirmed by means of a specific PCR technique developed in our lab, which is based on SMg sequencing (Appendix). Sanger sequencing of PCR products yielded a sequence identical to that generated by SMg. No circovirus sequence was found in the environmental control.

Conclusions
Our shotgun metagenomics approach enabled us to identify a putative new member of the Circoviridae family, provisionally named C. parisii, in a profoundly immunosuppressed patient who had self-resolving acute hepatitis. Phylogenetic analysis showed clustering of the new virus with members of the Circovirus genus known to infect different animal species. As for other circoviruses, the viral genome displayed an origin of replication (lacking 1 nucleotide), a replicase gene spanning 6 conserved regions, a capsid protein gene, and an ORF3, the role of which remains unknown.
Circoviruses are single-stranded DNA viruses generally transmitted via the fecal-oral route, with a potential pathogenic role in animals. Thus far, no human circovirus infections have been recorded (10), and serologic studies have not revealed any human contact (11). Nevertheless, culture of Porcine circovirus 2 on human cell lines, including liver cells, demonstrates the ability of this virus to replicate in human cells (12). Various pathologies have been observed in animals infected with circoviruses, including hepatitis (13,14). Porcine circovirus 3, the closest known circovirus, causes respiratory and neurologic diseases, cardiac and multisystemic inflammation, reproductive failure, Figure. Genomic and phylogenetic analysis of putative novel virus, Circovirus parisii, from an immunocompromised patient with hepatitis, France, 2022. A) Full-length genome of C. parisii reconstructed from shotgun metagenomics (SMg) sequence analysis. The genome is a 2021-nt singlestranded circular DNA containing 3 predicted open reading frames (ORFs), including ORF1 (replicase, green), ORF2 (capsid protein, blue) and ORF3 (red). The stem-loop contains an AGTATTAC sequence (origin of replication) that misses 1 nt (red dash) compared with other circoviruses. An ATG start codon is located at the 5′ end of the replicase gene. The replicase gene contains 6 conserved motifs, represented with a yellow background (amino acid and nucleotide sequences), with silent substitutions in red. B) Phylogenetic analysis of the replicase gene of the Circoviridae family, including the newly discovered C. parisii (red dot), known circoviruses (blue), and known cycloviruses (pink). Bootstrap values >70% are indicated with black stars. Scale bar indicates substitutions per site. and porcine dermatitis and nephropathy syndrome (15). The presence of the novel virus at the time of the aminotransferase peak raises questions about the causal relationship. Other techniques, such as in situ hybridization on infected tissue, might have offered some insights but were not available in our case. The source of transmission-perhaps animal, perhaps humancould not be established based on this patient's history.

About the Author
Dr. Rodriguez is a professor at Assistance Publique-Hôpitaux de Paris, University Paris-Est Créteil, INSERM U955 Team 18. His research interests are infectious diseases, metagenomics, diagnostic, transcriptomics, virology, and emerging pathogens.